Withania somnifera (L.) Dunal (Ashwagandha); current understanding and future prospect as a potential drug candidate.

Cancer and Neurodegenerative diseases are one of the most dreadful diseases to cure and chemotherapy has found a prime place in cancerous treatments while as different strategies have been tested in neurodegenerative diseases as well. However, due to adverse shortcomings like the resistance of cancerous cells and inefficiency in neurodegenerative disease, plant sources have always found a prime importance in medicinal use for decades, Withania somnifera (L.) Dunal (W. somnifera) is a well-known plant with medicinal use reported for centuries. It is commonly known as winter cherry or ashwagandha and is a prime source of pharmaceutically active compounds withanolides. In recent years research is being carried in understanding the extensive role of W. somnifera in cancer and neurological disorders. W. somnifera has been reported to be beneficial in DNA repair mechanisms; it is known for its cellular repairing properties and helps to prevent the apoptosis of normal cells. This review summarizes the potential properties and medicinal benefits of W. somnifera especially in cancer and neurodegenerative diseases. Available data suggest that W. somnifera is effective in controlling disease progressions and could be a potential therapeutic target benefiting human health status. The current review also discusses the traditional medicinal applications of W. somnifera, the experimental evidence supporting its therapeutical potential as well as obstacles that necessitate being overcome for W. somnifera to be evaluated as a curative agent in humans.

Adapalene inhibits the growth of triple-negative breast cancer cells by S-phase arrest and potentiates the antitumor efficacy of GDC-0941.

Although advances in diagnostics and therapeutics have prolonged the survival of triple-negative breast cancer (TNBC) patients, metastasis, therapeutic resistance, and lack of targeted therapies remain the foremost hurdle in the effective management of TNBC. Thus, evaluation of new therapeutic agents and their efficacy in combination therapy is urgently needed. The third-generation retinoid adapalene (ADA) has potent antitumor activity, and using ADA in combination with existing therapeutic regimens may improve the effectiveness and minimize the toxicities and drug resistance. The current study aimed to assess the anticancer efficacy of adapalene as a combination regimen with the PI3K inhibitor (GDC-0941) in TNBC in vitro models. The Chou-Talalay's method evaluated the pharmacodynamic interactions (synergism, antagonism, or additivity) of binary drug combinations. Flow cytometry, Western blotting, and in silico studies were used to analyze the mechanism of GDC-ADA synergistic interactions in TNBC cells. The combination of GDC and ADA demonstrated a synergistic effect in inhibiting proliferation, migration, and colony formation of tumor cells. Accumulation of reactive oxygen species upon co-treatment with GDC and ADA promoted apoptosis and enhanced sensitivity to GDC in TNBC cells. The findings indicate that ADA is a promising therapeutic agent in treating advanced BC tumors and enhance sensitivity to GDC in inhibiting tumor growth in TNBC models while reducing therapeutic resistance.

Folic Acid Levels During Pregnancy Regulate Trophoblast Invasive Behavior and the Possible Development of Preeclampsia.

Background: One of the unique features of placentation is its similarity to tumorigenesis yet being very well regulated. It allows rapid proliferation, migration, and invasion of mononuclear trophoblast cells into the maternal uterus and remodeling the maternal vasculature. This pseudomalignant nature of trophoblastic cells is strictly regulated and its importance becomes evident in abnormal pregnancies that are characterized by aberrant trophoblast proliferation/invasion like preeclampsia. In addition to this, the importance of folic acid supplementation during pregnancy is well documented. We aimed to analyze the molecular and epigenetic regulation of the pseudomalignant nature of placentation via folic acid levels. Methods: Placental tissue samples were collected from different pregnancies in three different gestational stages. We estimated the impact of folic acid levels on global methylation, LINE1 methylation, and expression of DNMTs in all three gestational stages in pregnant women and preeclampsia pregnancies. We also analyzed the effect of folic acid supplementation on trophoblastic invasion using placental derived cells viz, JEG-3 and HTR-8/SVneo cell line and verified the molecular and epigenetic mechanisms involved in this regulation. Results: Development of preeclampsia was observed to be associated with lower folate levels in placental tissue, higher global methylation level, and higher expression of DNMT1and DNMT3A. Folic acid supplementation was found to increase the invasive potential of placental trophoblasts by almost two folds which were associated with the decreased expression of tumor suppressor genes and tissue inhibitors of matrix metalloproteinases; and increased expression of oncogenes, telomerase gene, and matrix metalloproteinases. These folic acid-mediated changes were observed to be regulated by CpG methylation in the case of many genes. Folic acid supplementation was also observed to significantly decrease global methylation in placental trophoblasts related to decreasing expression of DNMT1 and DNMT3A. Conclusion: Lower folic acid levels are associated with preeclampsia development and folic acid supplementation regulates the invasive potential of placental trophoblasts as mediated by various epigenetic changes in the placenta suggesting the protective effect of folic acid against preeclampsia.

Chemokines in triple-negative breast cancer heterogeneity: New challenges for clinical implications.

Tumor heterogeneity is a hallmark of cancer and one of the primary causes of resistance to therapies. Triple-negative breast cancer (TNBC), which accounts for 15-20% of all breast cancers and is the most aggressive subtype, is very diverse, connected to metastatic potential and response to therapy. It is a very diverse disease at the molecular, pathologic, and clinical levels. TNBC is substantially more likely to recur and has a worse overall survival rate following diagnosis than other breast cancer subtypes. Chemokines, low molecular weight proteins that stimulate chemotaxis, have been shown to control the cues responsible for TNBC heterogeneity. In this review, we have focused on tumor heterogeneity and the role of chemokines in modulating tumor heterogeneity, since this is the most critical issue in treating TNBC. Additionally, we examined numerous cues mediated by chemokine networks that contribute to the heterogeneity of TNBC. Recent developments in our knowledge of the chemokine networks that regulate TNBC heterogeneity may pave the way for developing effective therapeutic modalities for effective treatment of TNBC.

The tumor microenvironment as driver of stemness and therapeutic resistance in breast cancer: New challenges and therapeutic opportunities.

BACKGROUND: Breast cancer (BC), the second most common cause of cancer-related deaths, remains a significant threat to the health and wellness of women worldwide. The tumor microenvironment (TME), comprising cellular components, such as cancer-associated fibroblasts (CAFs), immune cells, endothelial cells and adipocytes, and noncellular components such as extracellular matrix (ECM), has been recognized as a critical contributor to the development and progression of BC. The interplay between TME components and cancer cells promotes phenotypic heterogeneity, cell plasticity and cancer cell stemness that impart tumor dormancy, enhanced invasion and metastasis, and the development of therapeutic resistance. While most previous studies have focused on targeting cancer cells with a dismal prognosis, novel therapies targeting stromal components are currently being evaluated in preclinical and clinical studies, and are already showing improved efficacies. As such, they may offer better means to eliminate the disease effectively. CONCLUSIONS: In this review, we focus on the evolving concept of the TME as a key player regulating tumor growth, metastasis, stemness, and the development of therapeutic resistance. Despite significant advances over the last decade, several clinical trials focusing on the TME have failed to demonstrate promising effectiveness in cancer patients. To expedite clinical efficacy of TME-directed therapies, a deeper understanding of the TME is of utmost importance. Secondly, the efficacy of TME-directed therapies when used alone or in combination with chemo- or radiotherapy, and the tumor stage needs to be studied. Likewise, identifying molecular signatures and biomarkers indicating the type of TME will help in determining precise TME-directed therapies.

Dimethoate Induces DNA Damage and Mitochondrial Dysfunction Triggering Apoptosis in Rat Bone-Marrow and Peripheral Blood Cells.

Dimethoate (DM) is an organophosphorus (OP) pesticide with wide use in the pest control. Its persistence in crops and soils could possibly cause adverse health consequences in humans as well as other non-target species. Since molecular studies confirming potential genotoxicity of DM have not been previously reported, the acute in vivo toxicological impact was evaluated in Wistar rats. Significant micronuclei induction and metaphase chromosome abnormalities in bone marrow cells exposed to three different DM doses (20, 40 and 60 mg/kg-bw) at multiple treatment durations (24, 48 and 72 h) indicated positive dose response relationship, confirming its genotoxic and cytotoxic potential. Significant mitotic index decrease was seen in dosed animals compared to vehicle control. The study used peripheral blood comet assay, indicating DM-mediated damage to DNA at all exposure levels in a time responsive manner. These assays were found to be an effective, precise, and fast technique with applied value in biomonitoring studies. Cell cycle and apoptosis along with mitochondrial membrane potential (MMP) in flow cytometric analyses confirmed DM exposure decreased MMP, affected the cell cycle, and inflicted DNA damage, which led to cellular apoptosis of leukocytes culminating into immunotoxic effects. The in silico experiments consequently augmented that DM showed acceptable binding energy value for Cyclin A2, suggesting that it could inhibit the cell cycle progression by inhibiting cyclin A2.

Recent Advances in Head and Neck Tumor Microenvironment-Based Therapy.

Head and neck squamous cell carcinomas (HNSCCs) are a group of heterogeneous aggressive tumors affecting more than half a million patients worldwide annually. While the tobacco- and alcohol-associated HNSCC tumors are declining, human papillomavirus (HPV)-induced tumors are on rise. Despite recent advances in multimodality therapeutic interventions including surgery in combination with chemoradiation therapy (CRT), the overall 5-year survival has not improved more than 50%. The underlying reasons for this dismal prognosis is the intrinsic or acquired resistance to CRT. While previous studies were focused to target tumor cells, recent findings have implicated the involvement of tumor microenvironment (TME) on tumor progression and response to therapy. HNSCC TME includes cancer-associated fibroblasts (CAFs), endothelial cells, immune cells, endocrine cells, and the extracellular matrix (ECM) proteins including collagen and fibronectin. Understanding the crosstalk between TME and cancer cells is important to formulate more effective novel therapies and to overcome resistance mechanisms. Here, we summarized the current literature on recent advances on HNSCC TME with special emphasis on novel cell-cell interactions and therapies currently under development.

Dihydropyrimidinones: efficient one-pot green synthesis using Montmorillonite-KSF and evaluation of their cytotoxic activity.

A simple, efficient, cost-effective, recyclable and green approach has been developed for the synthesis of new dihydropyrimidinone analogs via the Biginelli reaction. The methodology involves a multicomponent reaction catalyzed by "HPA-Montmorillonite-KSF" as a reusable and heterogeneous catalyst. This method gives an efficient and much improved modification of the original Biginelli reaction, in terms of yield and short reaction times under solvent free conditions. All the derivatives were subjected to cytotoxicity screening against a panel of four different human cancer cell lines viz. colon (Colo-205), prostate (PC-3), leukemia (THP-1) and lung (A549) to check their effect on percentage growth. MTT [3-(4,5-dimethylthiazol-yl)-diphenyl tetrazoliumbromide] cytotoxicity assay was employed to check IC50 values. Of the synthesized analogs, 16a showed the best activity with IC50 of 7.1 ± 0.8, 13.1 ± 1.4, 13.8 ± 0.9 and 14.7 ± 1.1 µM against lung (A549), leukemia (THP-1), prostate (PC-3) and colon (Colo-205) cancer lines, respectively. The 16a analog was further checked for its effect on cancer cell properties through clonogenic (colony formation) and scratch motility (wound healing) assays and thereby was found that it reduced both the colony formation and migratory properties of the lung cancer cell line (A549). Further, molecular docking studies were performed with 16a to show its binding mode.

An anti-cancerous protein fraction from Withania somnifera induces ROS-dependent mitochondria-mediated apoptosis in human MDA-MB-231 breast cancer cells.

Withania somnifera exhibits different pharmacological activities which mainly stem from its broad range of bioactive molecules. Majority of these bioactive molecules, fall into the groupings of alkaloids, steroidal lactones, phenolic compounds and glycoproteins. In this study, we evaluated a novel protein fraction, named here as WSPF, isolated from Withania somnifera roots for its cytotoxic properties against various human cancer cell lines. WSPF exhibited apoptotic activity for each cancer cell line tested, demonstrating significant activity against MDA-MB-231 human breast cancer cells with an IC50 value of 92 µg/mL. WSPF induced mitochondrial-mediated apoptosis of MDA-MB-231 cells via extensive reactive oxygen species generation, dysregulation of Bax/Bcl-2, loss of mitochondrial membrane potential and caspase-3 activation. Additionally, we observed G2/M-phase cell cycle arrest, cleavage of nuclear lamin A/C proteins, and nuclear morphological changes. The present results highlight the anti-cancer properties of WSPF, indicating that the proteins in this fraction can be potential therapeutic agents for triple negative breast cancer treatment.

Synthesis and Biological Evaluation of Novel Osthol Derivatives as Potent Cytotoxic Agents.

BACKGROUND: Natural product, osthol has been found to have important biological and pharmacological roles particularly having inhibitory effect on multiple types of cancer. OBJECTIVE: The unmet needs in cancer therapeutics make its derivatization an important and exciting field of research. Keeping this in view, a whole new series of diverse analogues of osthol (1) were synthesized. METHOD: All the newly synthesized compounds were made through modification in the lactone ring as well as in the side chain of the osthol molecule and were subjected to anti-proliferative screening through 3-(4,5-Dimethylthiazol-yl)-diphenyl tetrazoliumbromide (MTT) against four different human cancers of diverse origins viz. Colon (Colo-205), lung (A549), Leukemia (THP- 1) and breast (MCF-7) including SV40 transformed normal breast epithelial cell (fR-2). RESULTS: Interestingly, among the tested molecules, most of the analogs displayed better antiproliferative activity than the parent Osthol 1. However, among all the tested analogs, compound 28 exhibited the best results against leukemia (THP1) cell line with IC50 of 5µM.Compound 28 induced potent apoptotic effects and G1 phase arrest in leukemia cancer cells (THP1). The population of apoptotic cells increased from 13.8% in negative control to 26.9% at 8µM concentration of 28. Compound 28 also induced a remarkable decrease in mitochondrial membrane potential (ΛΨm) leading to apoptosis of the cancer cells. CONCLUSION: A novel series of molecules derived from natural product osthol were synthesized, wherein compound 28 was found to be most effective against leukemia and with 10 fold less toxicity against normal cells. The compound induced cancer inhibition mainly through apoptosis and thus has a potential in cancer therapeutics.

Proproliferative function of adaptor protein GRB10 in prostate carcinoma.

Growth factor receptor-binding protein 10 (GRB10) is a well-known adaptor protein and a recently identified substrate of the mammalian target of rapamycin (mTOR). Depletion of GRB10 increases insulin sensitivity and overexpression suppresses PI3K/Akt signaling. Because the major reason for the limited efficacy of PI3K/Akt-targeted therapies in prostate cancer (PCa) is loss of mTOR-regulated feedback suppression, it is therefore important to assess the functional importance and regulation of GRB10 under these conditions. On the basis of these background observations, we explored the status and functional impact of GRB10 in PCa and found maximum expression in phosphatase and tensin homolog (PTEN)-deficient PCa. In human PCa samples, GRB10 inversely correlated with PTEN and positively correlated with pAKT levels. Knockdown of GRB10 in nontumorigenic PTEN null mouse embryonic fibroblasts and tumorigenic PCa cell lines reduced Akt phosphorylation and selectively activated a panel of receptor tyrosine kinases. Similarly, overexpression of GRB10 in PTEN wild-type PCa cell lines accelerated tumorigenesis and induced Akt phosphorylation. In PTEN wild-type PCa, GRB10 overexpression promoted mediated PTEN interaction and degradation. PI3K (but not mTOR) inhibitors reduced GRB10 expression, suggesting primarily PI3K-driven regulation of GRB10. In summary, our results suggest that GRB10 acts as a major downstream effector of PI3K and has tumor-promoting effects in prostate cancer.-Khan, M. I., Al Johani, A., Hamid, A., Ateeq, B., Manzar, N., Adhami, V. M., Lall, R. K., Rath, S., Sechi, M., Siddiqui, I. A., Choudhry, H., Zamzami, M. A., Havighurst, T. C., Huang, W., Ntambi, J. M., Mukhtar, H. Proproliferatve function of adaptor protein GRB10 in prostate carcinoma.

AKT Inhibition Modulates H3K4 Demethylase Levels in PTEN-Null Prostate Cancer.

Hyperactivated AKT kinase due to loss of its negative regulator PTEN influences many aspects of cancer biology, including chromatin. AKT primarily regulates acetyl-CoA production and phosphorylates many histone-modulating enzymes, resulting in their activation or inhibition. Therefore, understanding the therapeutic impact of AKT inhibition on chromatin-related events is essential. Here, we report that AKT inhibition in prostate-specific PTEN knockout mice significantly induces di- and trimethylation of H3K4 with concomitant reduction in H3K9 acetylation. Mechanistically, we observed that AKT inhibition reduces expression of the H3K4 methylation-specific histone demethylases KDM5 family, especially KDM5B expression at transcriptional levels. Furthermore, we observed that AKT negatively regulates miR-137 levels, which transcriptionally represses KDM5B expression. Overexpression of miR-137 significantly reduced KDM5B and increased H3K4 methylation levels but failed to change AKT phosphorylation. Overall, we observed that AKT transcriptionally regulates KDM5B mainly via repression of miR-137. Our data identify a mechanism by which AKT kinase modulates the prostate cancer epigenome through regulating H3K4 methylation. Additional studies on AKT inhibition-mediated induction of H3K4 methylation will help in designing strategies to enhance the therapeutic efficacy of PI3K/AKT inhibitors.

Synthesis and Biological Evaluation of Novel Triazoles Linked 7-hydroxycoumarin as Potent Cytotoxic Agents.

BACKGROUND: BacCancer is regarded as second leading cause of death worldwide. Therefore, there is a high demand for the discovery, development and improvement of novel anti-cancer agents which could efficiently prevent proliferative pathways and clonal expansion of cells. OBJECTIVE: In view of this, a new series of bioactive scaffolds viz triazoles linked 7-hydroxycoumarin (1) were synthesized using click chemistry approach. METHOD: All the synthesized compounds were screened for cytotoxicity against a panel of seven different human cancer cell lines viz. Colon (Colo-205 and HCT-116), breast (MCF-7), lung (NCI-H322 and A549), prostate (PC-3) and skin (A-431) using 3-(4,5-Dimethylthiazol-yl)-diphenyl tetrazoliumbromide (MTT) assay. RESULTS: Among all tested analogs, compound 5, displayed better cytotoxic activity as compared to the parent 7- hydroxycoumarin (1) with IC50 of 5.1, 22.7, 14.3 and 10.2 µM against breast (MCF-7), lung (NCI- H322), prostate (PC-3) and skin (A-431) cancer cell lines, respectively; the compound 5 was 8-fold more sensitive against MCF-7 than the parent 7-hydroxycoumarin. Moreover, Compound 5 induced both cytotoxic as well as cytostatic effects via induction of apoptosis and G1 phase arrest, respectively in breast cancer cells (MCF-7). The apoptotic cell population enhanced to 18.8% at 8 µM of 5 from 9.8% in case of negative control, while G1 phase arrest increased to 54.4% at 8 µM compared to negative control of 48.1%. Moreover, Compound 5 also exhibited a remarkable decrease in mitochondrial membrane potential (ΛΨm) leading to apoptosis of cancer cells used. CONCLUSION: The structure-activity relationship study revealed that the derivatives bearing electron-withdrawing substituents were more effective. The present study resulted in identification of the compounds demonstrating broad spectrum cytotoxic activity.

Attenuation of Glutamate-Induced Excitotoxicity by Withanolide-A in Neuron-Like Cells: Role for PI3K/Akt/MAPK Signaling Pathway.

Glutamate-induced excitotoxicity is one of the major underlying mechanisms for neurodegenerative diseases. Efforts are being made to treat such conditions with an array of natural compounds that can modulate the release of glutamate or the underlying mechanisms associated with it. Withania somnifera extract has potent pharmacologic activity similar to that of Korean Ginseng tea and is used to treat several neuronal disorders. However, to date, little efforts have been made to evaluate individual constituents of this plant for neurodegenerative disorders. Present study was carried out to investigate withanolide-A, one of the active constituents of Withania somnifera against glutamate-induced excitotoxicity in retinoic acid differentiated Neuro2a neuroblastoma cells. The results indicated that glutamate treatment for 2 h induced death in cells that was significantly attenuated by pre-treatment with MK-801 (specific NMDA receptor antagonist) and different concentrations of withanolide-A. Withanolide-A abated the glutamate-induced influx of intracellular calcium and excessive ROS production significantly. Further on, glutamate treatment resulted in increased levels of pro-apoptotic and decreased levels of anti-apoptotic proteins, and these protein levels were normalized by various doses of withanolide-A. All of these protective effects were partly due to inhibition of MAPK family proteins and activation of PI3K/Akt signaling. Thus, our results suggest that withanolide-A may serve as potential neuroprotective agent.

Modulation of dietary folate with age confers selective hepatocellular epigenetic imprints through DNA methylation.

The present study has been designed to determine the effect of folate modulation (deficiency/supplementation) with aging on the promoter methylation of tumor suppressor and proto-oncogenes to understand the underlying mechanism of epigenetic alterations. Folate deficiency was induced for 3 and 5 months in weanling, young and adult groups, and after 3 months of folate deficiency, they were repleted with physiological folate (2 mg/kg diet) and folate oversupplementation (8 mg/kg diet) for another 2 months. The methylation facet in the present study revealed that the combined effect of folate deficiency and aging decreased the methylation index. Folate deficiency with age resulted in the up-regulation of proto-oncogenes (C-MYC and C-JUN) and cell cycle regulator gene Cyclin E as a result of promoter hypomethylation. However, in case of tumor suppressor genes (p53, p15ink4b and p16ink4a), the expression levels were found to be decreased at transcriptional level due to promoter hypermethylation. Upon repletion with physiological folate and folate oversupplementation, we found down-regulation of proto-oncogenes and up-regulation of tumor suppressor genes as a result of promoter hypermethylation and hypomethylation, respectively. Deregulation of these important genes due to folate deficiency may contribute toward the pathogenesis at cellular level.

Immunostimulatory activity of plumieride an iridoid in augmenting immune system by targeting Th-1 pathway in balb/c mice.

Plumieride, an iridoid glucoside isolated from Plumieria acutifolia leaves was investigated for its immunostimulatory activity on humoral, cell mediated and intracellular cytokine levels in sensitized and unsensitised balb/c mice. Plumieride restores the suppressed cell mediated, humoral immune response and also enhances the release of TNF- α, IFN-γ, and IL-2 (Th-1) in immune compromised cyclosporine and cyclophosphamide treated balb/c mice. It does not stimulate the IL-4 (Th-2) expression. Plumieride demonstrates significant augmentation of Th-1 response in immunosuppressed balb/c mice. Results of the present study suggested that plumieride can be developed as an immunostimulatory adjuvant to treat the immune suppression in various disease condition(s).

Exploring Derivatives of Quinazoline Alkaloid l-Vasicine as Cap Groups in the Design and Biological Mechanistic Evaluation of Novel Antitumor Histone Deacetylase Inhibitors.

l-Vasicine is a quinazoline alkaloid with an electron dense ring and additional functionalities in its structure. Employing target oriented synthesis (TOS) based on in silico studies, molecules with significant docking scores containing different derivatives of l-vasicine as caps were synthesized. Interestingly, one molecule, i.e., 4a, which contained 3-hyroxypyrrolidine as a cap group and a six carbon long aliphatic chain as a linker was found to inhibit HDACs. 4a showed more specificity toward class I HDAC isoforms. Also 4a was found to be less cytotoxic toward normal cell lines as compared to cancer cell lines. 4a inhibited cancer cell growth and induced cell death by various mechanisms. However, 4a was found to induce cell death independent of ROS generation, and unlike many natural product based HDAC inhibitors, 4a was found to be nontoxic under in vivo conditions. Importantly, we for the first time report the possibility of using a 3-hydroxypyrrolidine cap for the synthesis of HDAC inhibitors with good potency.

Epigenetic modifications at DMRs of placental genes are subjected to variations in normal gestation, pathological conditions and folate supplementation.

Invasive placentation and cancer development shares many similar molecular and epigenetic pathways. Paternally expressed, growth promoting genes (SNRPN, PEG10 and MEST) which are known to play crucial role in tumorogenesis, are not well studied during placentation. This study reports for the first time of the impact of gestational-age, pathological conditions and folic acid supplementation on dynamic nature of DNA and histone methylation present at their differentially methylated regions (DMRs). Here, we reported the association between low DNA methylation/H3K27me3 and higher expression of SNRPN, PEG10 and MEST in highly proliferating normal early gestational placenta. Molar and preeclamptic placental villi, exhibited aberrant changes in methylation levels at DMRs of these genes, leading to higher and lower expression of these genes, respectively, in reference to their respective control groups. Moreover, folate supplementation could induce gene specific changes in mRNA expression in placental cell lines. Further, MEST and SNRPN DMRs were observed to show the potential to act as novel fetal DNA markers in maternal plasma. Thus, variation in methylation levels at these DMRs regulate normal placentation and placental disorders. Additionally, the methylation at these DMRs might also be susceptible to folic acid supplementation and has the potential to be utilized in clinical diagnosis.

The role of aberrant methylation of trophoblastic stem cell origin in the pathogenesis and diagnosis of placental disorders.

OBJECTIVES: The objective of the study is to investigate the role of methylation levels at promoter regions of placental vascularization genes (VEGF, EGFR, and c-jun) in pathogenesis and diagnosis of placental disorders. METHODS: We analyzed DNA and histone methylation at promoters of VEGF, EGFR, and c-jun via methylation-sensitive high-resolution melting and chromatin immunoprecipitation assay in pregnant women with normal pregnancy in first, second, and third trimesters (n = 30 in each group) and pregnant women with pregnancy complicated with preeclampsia (n = 30) and hydatidiform mole (n = 15). RESULTS: The higher expression of VEGF, EGFR, and c-jun in early pregnancy was observed to be independent of DNA methylation, while it was associated with H3 K9/K27 trimethylations. Also, abnormally higher expression of c-jun in GTDs was associated with lower H3K9me3 level at its promoter. Under preeclampsia conditions, we observed dysregulation of both DNA methylation and H3 trimethylation and subsequent low expression of VEGF, EGFR, and c-jun. Importantly, our promoter methylation data indicated that VEGF may act as novel fetal DNA diagnostic marker for preeclampsia and molar pregnancies in maternal plasma. CONCLUSION: These findings emphasize the importance of dysregulated epigenetic phenomenon behind the pathologies of placental disorders and use of promoter region DNA methylation as an epigenetic marker for these pathological pregnancies. © 2016 John Wiley & Sons, Ltd.

Withanone, an Active Constituent from Withania somnifera, Affords Protection Against NMDA-Induced Excitotoxicity in Neuron-Like Cells.

Withania somnifera has immense pharmacologic and clinical uses. Owing to its similar pharmacologic activity as that of Korean Ginseng tea, it is popularly called as Indian ginseng. In most cases, extracts of this plant have been evaluated against various diseases or models of disease. However, little efforts have been made to evaluate individual constituents of this plant for neurodegenerative disorders. Present study was carried out to evaluate Withanone, one of the active constituents of Withania somnifera against NMDA-induced excitotoxicity in retinoic acid, differentiated Neuro2a cells. Cells were pre-treated with 5, 10 and 20 µM doses of Withanone and then exposed to 3-mM NMDA for 1 h. MK801, a specific NMDA receptor antagonist, was used as positive control. The results indicated that NMDA induces significant death of cells by accumulation of intracellular Ca2+, generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, crashing of Bax/Bcl-2 ratio, release of cytochrome c, increased caspase expression, induction of lipid peroxidation as measured by malondialdehyde levels and cleavage of poly(ADP-ribose) polymerase-1 (Parp-1), which is indicative of DNA damage. All these parameters were attenuated with various doses of Withanone pre-treatment. These results suggest that Withanone may serve as potential neuroprotective agent.